In general, urologists tend to think that lower urinary tract symptoms (LUTS) make it easy to recall voiding and storage symptoms [1]. The effect of international prostate symptom score (IPSS), which is often used for LUTS treatment, is thought to have a lot of influence, but poorly treated post micturition dribble (PMD) is likely to escape the attention of urologists [2]. PMD is a term used to describe the involuntary loss of urine immediately after an individual finishes passing urine, usually after leaving the toilet in men or after rising from the toilet in women [3]. PMD is one of the most bothersome symptoms of middle-aged male LUTS, however the etiology and treatment mechanism of PMD differ from those of conventional lower urinary tract symptoms, so proper treatment is not done yet [1, 2]. Factors associated with PMD include weakening of the bulbocavernosus muscle, an abnormal urethro-corporocavernosal reflex, reduced compliance of the bulbar urethra, weakening of the external urethral sphincter, and bladder neck obstruction [4]. Several pathophysiology has been proposed, so the mechanisms we noted are the weakening of the bulbocavernouos muscle and the dilation of urethra due to atrophy of cavernosal tissue [1, 3].
Although clinical trial results have already been published, there are no preclinical studies on the evidence. So, we want to develop a disease animal model for PMD and compare the effects of senile change of penis and urethra. So the purpose of this study is to investigate the mechanism of PMD by observing the anatomical changes of the corpus cavernosum and urethra.
This study was performed in accordance with the Guide for Care and Use of Laboratory Animals.
All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea (CUMC-2016-0218-01).
Experimental animals were obtained from Orient Bio Co. (Seongnam, Korea) 24 months old male Sprague-Dawley rats were chosen for the old rat group and 8-week-old male Sprague-Dawley rats were chosen as a young rat group. All rats were sacrificed and then whole urethra from prostate to urethral orifice and corpus cavernosum were collected for histochemical and Western blot analysis.
Sections were viewed using an Carl Zeiss LSM900 w/AiryscanII Confocal microscope with ×20 objectives. Digital images were obtained using a Zeiss LSM 900 confocal microscope (Zeiss, Oberkochen, Germany), smooth muscle ratio and urethral luminal area was calculated using AxioVision Rel. 4.8 Software (Carl Zeiss Kore, Seoul, South Korea).
A 23-gauge butterfly needle containing 250 U/mL heparin solution was carefully inserted into the proximal corpus cavernosum and the other end of the PE-50 tube was connected to a pressure transducer (Grass model S48 K; Astro-Med Inc., West Warwick, RI, USA), to measure the ICP [5]. A bipolar stainless-steel electrical stimulator was used to stimulate the major pelvic ganglion at 10 V for 100 s and 2.4 mA with a pulse width of 2.5 ms. The maximum ICP value of three stimulations was used for the statistical analysis in each rat. ICP was normalized to MAP, which was recorded using a BD Intramedic PE-50 tubing (BD, Franklin Lakes, NJ, USA) inserted into the left carotid artery at the same time [6].
The collected tissue samples were fixed in 4% parafor-maldehyde for 24 h at 4℃ before creating a paraffin block. The following primary antibodies were used: nNOS (diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), α-SMA (diluted 1:500; Abcam, Cambridge, UK), and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 800 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).
Each set of sections was processed with Masson’s trichrome staining for urethral muscle fiber and collagen components. Urethral tissues were immersed in warm (60℃) Bouin solution for 30 minutes, rinsed, then stained with Weigert Hematoxylin for 10 minutes, and then rinsed until only nuclei remained stained. The sections were then stained with Biebrich Scarlet-Acid Fuchsin for 3 minutes, rinsed, and immersed in phosphomolybdic acid for 40 minutes. Next, the sections was stained with Aniline Blue for 5 minutes, rinsed in distilled water for 3 minutes, immersed in 1% acetic acid for 1 minutes, Finally, the sections were dehydrated through increasing concentrations of ethanol, and mounted.
The collected tissue was homogenized using ice-cold RIPA buffer (Cell Signaling Technology) containing ethylene diamine tetra acetic acid-free protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland) and particulate mass was removed by centrifugation (15,000×
Tissues were treated with 350 mL of 0.1 M HCl, after which silica beads were added (BioSec Enviro, Inc., Guelph, ON, Canada). The resulting samples were processed through a homogenizer (Precellys 24; Bertin Technologies, Montigny-le-Bretonneux, France) and spun by centrifugation at 12,000×g for 10 minutes at 4℃, after which their supernatants were extracted. A cyclic guanosine monophosphate (cGMP) direct immu-noassay kit (K372-100; BioVision, Mountain View, CA, USA) was used to measure corporal cGMP levels.
All data are presented as means±standard error (SD) and were analyzed using SPSS version 22.0 (IBM, Armonk, NY, USA). Student’s
The diameter of bulbous urethra was statistically signi-ficantly greater in old rats than in young rats (
Measurement of diameter and area of bulbous urethra and corpus cavernosum
Bulbous urethra | Corpus cavernosum | Urethra/ total penis |
Corpus cavernosum/total penis | |
---|---|---|---|---|
Young (µm2) | 191049.66±1235.56 | 3286741±6781.73 | 2.42±1.54% | 46.10±4.29% |
Old (µm2) | 538470±199.11 | 5259875±2175.63 | 4.22±0.16% | 39.69±1.75% |
<0.001 | <0.001 | <0.001 | <0.01 |
All rats were tested for erectile function by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) under anesthesia. Representative images of ICP results are shown in Figure 1. The old group showed statistically significantly lower ICP values than young group. In a quantitative analysis (Figure 1B), the ICP of the young group was 0.82±0.11, and the old group was 0.28±0.07. The mean ratio was significantly lower in the old group than in the young group (
Masson’s trichrome staining was performed to visualize the urethra present at the site. The smooth muscle and collagen contents in the urethra were observed by Masson’s trichrome staining. Figure 2A illustrates images of transverse sections of the proximal and the distal urethra, which gradually decreased in size (Figure 2B). In the young distal urethra the lumen is crescent‐shaped due to the presence of the smooth muscle protrusion. As shown in Figure 2, the smooth muscle contents were higher in the young group than in the old group (
In the urethra, smooth muscle positive areas were analyzed by immunohistochemical staining (Figure 3A). As shown in Figure 3, α-smooth muscle actin (α-SMA) expression levels were elevated in the urethra, indicating that smooth muscle higher in young rat urethra tissues (
In the corpus cavernosum, smooth muscle positive areas were analyzed by immunohistochemical staining (Figure 4A). As shown in Figure 4, α-SMA expression levels were elevated in the corpus cavernosum, indicating that smooth muscle higher in young rat tissues (
Representative images of nNOS staining in the copus carvernosum is shown nNOS was lower in the old group than in the young group (Figure 5A). Immunostaining for neuronal nitric oxide synthase (nNOS) (red) and immu-nostaining staining using nerve marker β-III tubulin (green) in the dorsal penile nerve. In a quantitative analysis, intensities were higher in the young group than in the old group (
As shown in Figure 6, in the young group, p-AKT/AKT levels were higher (
PMD is one of the most common symptoms of LUTS and big bothersome disease for men. Compared to this discomfort, the treatment and mechanism are not clear, so it can be regarded as one of the types of intractable diseases [7, 8]. The symptoms are not life-threatening and the response to treatment is insignificant, so research on this has not been conducted much recently [9].
In men, PMD is known to occur when urine collects in the bulbous urethra, but opinions are still controversial on the exact mechanism [10]. One of the purpose considered as a mechanism is that the urine collected in the urethra cannot be sufficiently emptied due to the weakening of the bulbocavernous muscle. It is also known that poor milk back of bulbous urethra occurs after end of voiding due to incomplete external urethral sphincter or bladder neck obstruction [11]. Our new hypothesis in addition to the previously mentioned mechanism, that the decrease in the volume of corpus cavernosum according to the aging process can cause the relative volume increase of the bulbous urethra to promote PMD.
So, we conducted a preclinical study using animals to prove this hypothesis. The factors we mainly wanted to report were the difference in urethral diameter by section and volume change of cavernosal tissue between young and old rats. In the previous studies, there have been many reports of senile changes in cavernosal tissue [12, 13]. Most of the experimental results reported an increase in fibrotic tissue and a decrease in smooth muscle as aging progressed in cavernosum. However, no animal studies have been found related to urethra to study PMD [14, 15]. Among the pathophysiology of PMD, our study was the first study conducted to prove our hypothesis that we confirmed the increase of bulbous urethral volume in old rats and analyzed the mechanism.
We identified p-AKT/AKT levels, bax, and bcl-2 levels in urethra and corpus cavernosum. In conclusion, it was confirmed that apoptosis in old rats increased statistically significant in urethra, and corpus cavernosum both. There are already many studies on the mecha-nism of erectile dysfunction due to apoptosis-induced reduction of smooth muscle and increase in collagen in corpus cavernosum [16-20]. Previous study has shown that eNOS elevated the cGMP level, which stimulated penile erection [21]. p-AKT could stimulate endothelial cells to produce eNOS, which is the factor for vasodilatation. It is possible that high levels endothelial cells thereby stimulate vasodilatation and inhibit smooth muscle atrophy [22]. Our results also showed that elevated levels of eNOS in the young group may be related to increased nNOS, higher cGMP levels, and reduced atrophy of the urethra smooth muscle. In the young group related to expression nNOS and cGMP levels, and reduced apoptosis of the corpus caver-nosum, as demonstrated by the increased β-III tubulin and nNOS expression. As a result of our study, this has an effect on the decrease in the volume of the corpus cavernosum, and it is thought that it also affects the increase in the diameter of the bulbous urethra in particular because of the relatively empty space. In addition, a decrease in bulbocavernous muscle contents was also observed, and it was confirmed through the above experiment as one of the known pathophy-siology of PMD.
According to one study [7], there is a literature on the effect of PDE5-inhibitor on not only smooth muscle but also skeletal muscle, so additional interesting research results can be expected on external sphincter which is related to the pathophysiology of PMD. Second, there was a lot of difference in total penis size between young and old rats, it was unreasonable to interpret the results by direct diameter, so the ratio with total size was used. For more accurate results, it would be better to proceed at a time when the penis sizes of young and old rats become similar. Third, since PMD symptoms cannot be fully reproduced using animals, this is only an indirect aspect of the clinical outcome and cannot be directly verified clinical symptoms.
Already reported that the PDE5 inhibitor has an effect on the symptoms of PMD through clinical studies, confirming that our hypothesis is sufficiently possible [7, 11]. It is also believed that more scientific verification will be possible through the expansion of animal experi-ments conducted this time. With these additional experi-ments, we plan to continue our research by helping to improve symptoms of difficult-to-treat PMD and clarifying our understanding of the mechanisms.
According to the recent International continence society (ICS) guideline, the term PMD is thought to be replaced by the term “post voiding incontinence”. The reason that the term of PMD, which has not received attention for a long time, has changed to post voiding incontinence is likely to be due to the recent increase in new issues. Although the pathophysiology of PMD is very diverse, it seems that there is a clear connection with our hypothesis with cavernosal tissue. We observed a decrease in cavernosum volume and an increase in bulbous urethra diameter in old rats, which is thought to be related to apoptosis caused by senile change. We expect our study to be a background that can help patients suffering from PMD.
중년 이후 많은 환자들이 배뇨 후 요실금을 앓고 있지만 아직 적절한 치료가 이뤄지지 않고 있다. 몇 가지 병태생리학적 기전이 제안되었지만 우리가 주목한 기전은 구해면체근과 해면체 조직의 위축으로 인한 요도의 확장이었다. 본 연구의 목적은 해면체와 요도의 해부학적 변화를 관찰하여 배뇨 후 요실금의 기전을 규명하는 것으로 8주령의 SD 수컷 랫드 그룹과 24개월 이상 된 SD 수컷 랫드 그룹으로 나누었다. 전체 음경에 대한 구부요도 및 해면체 면적의 비율은 두 그룹 간 통계적으로 유의하였다(
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Jeon SH, Professor; Park MY, Professor.